Flow Regulation of 72-kD Collagenase IV (MMP-2) After Experimental Arterial Injury

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1 Flow Regulation of 72-kD Collagenase IV (MMP-2) After Experimental Arterial Injury Hisham S. Bassiouny, MD; Ruo H. Song, MD; Xue F. Hong, MD; Ashok Singh, PhD; Hrachya Kocharyan, MD, PhD; Seymour Glagov, MD Background MMP-2 plays a key role in basement membrane degradation and in the migration of proliferating smooth muscle cells after vascular injury. Because low flow and shear stress have been related to the localization and progression of intimal hyperplasia, we hypothesized that flow conditions modulate in vivo MMP-2 transcription and activity in a model of injury-induced intimal thickening. Methods and Results The right common carotid artery (CCA) was balloon-injured in 21 New Zealand White male rabbits. Flow was thereafter preserved (normal flow, n 7), reduced by partial outflow occlusion (low flow, n 7), or increased by ligation of the left CCA (high flow, n 7). In 15 other animals (controls without injury), flow was reduced (n 5), increased (n 5), or preserved (n 5). Mean blood flow and pressure in the right CCA were measured before and after flow modulation (day 0) and before the rabbits were killed (day 7). Northern analysis, gelatin-gel zymography, and fluorometric assays were performed on day 7 to determine MMP-2 mrna levels and activity in relation to flow and intimal thickening. Mean flow was reduced from 21 1 to7 1 ml/min (P 0.05) by outflow occlusion and increased to 31 2 ml/min (P 0.05) by ligation of the contralateral CCA. Blood pressure was not different between the flow groups. Hemodynamic parameters were similar for days 0 and 7 after flow modulation. In the injured right CCA, there was a 186% increase in MMP-2 mrna with normal flow (P 0.05), a 366% increase with low flow (P 0.005), and only a 38% increase with high flow (P 0.05) compared with the uninjured CCA with normal flow. In the uninjured CCA, MMP-2 mrna levels were increased by only 39% and 26% in the low- and high-flow groups, respectively, compared with normal-flow controls. The zymographic signal and quantitative fluorescent activity of gelatinase were markedly increased in both injured and uninjured CCAs subjected to low flow. Intimal thickening was observed after 1 week only in CCA segments with low flow and injury. Conclusions Hemodynamic forces such as low flow upregulate injury-induced MMP-2 mrna and appear to be more important in regulating MMP-2 activity than injury alone. This may facilitate migration of the smooth muscle cells and subsequent development of intimal thickening. (Circulation. 1998;98: ) Key Words: hemodynamics metalloproteinases hyperplasia Intimal hyperplasia is an arterial mural response known to occur after vascular intervention and with altered hemodynamics. As such, it represents a healing or an adaptive reaction designed to maintain structural integrity of the vessel wall and homeostasis. This response involves migration and proliferation of the medial SMCs in the subintimal space. 1,2 By necessity, basement membrane degradation and extracellular matrix remodeling are required to liberate the SMC from its surrounding scaffold. 3 MMPs are a family of zinc-dependent enzymes that are able to digest a wide range of extracellular matrix proteins and work in concert during development, 4 during atherogenesis, 5 and after vascular injury. 6,7 The MMP family includes the collagenases (MMP-1), which degrade structural types I to III collagens; the stromelysins (MMP-3), which have a broad substrate specificity (proteoglycans, laminin, fibronectin); and the type IV collagenases, also called gelatinases. The latter group includes 72-kD gelatinase A (MMP-2) and 92-kD gelatinase B (MMP-9), which degrade type IV collagen (a major component of the subendothelial basement membrane); gelatin from degraded collagen; and elastin. There is increasing evidence that expression and activation of 72-kD and/or 92-kD type IV collagenase is associated with SMC migration and proliferation in vitro In vivo administration of synthetic MMP inhibitors significantly reduces neointimal thickening after injury in rat carotid artery by decreasing SMC migration and proliferation. 7,11 Evidence has also accrued that hemodynamic factors, namely low flow and shear stress, are associated with the localization and progression of intimal hyperplasia The mechanisms by which these hemodynamic forces may modulate the SMC proliferative and migratory responses in vivo include induction of mitogenic cytokines, such as PDGF-B, or other mediators, such as MMPs. We therefore investigated Received December 2, 1997; revision received January 27, 1998; accepted January 28, From the Department of Surgery and Pathology, the University of Chicago, and the Hektoen Institute for Medical Research (A.S.), Chicago, Ill. Reprint requests to Hisham S. Bassiouny, MD, Department of Surgery, the University of Chicago, 5841 S Maryland Ave, MC 5028, Chicago, IL hbassiou@surgery.bsd.uchicago.edu 1998 American Heart Association, Inc. 157

2 158 Flow Regulation of MMP-2 Selected Abbreviations and Acronyms CCA common carotid artery MAP mean arterial pressure MMP matrix metalloproteinase PDGF platelet-derived growth factor rrna ribosomal RNA SMC smooth muscle cell VSMC vascular smooth muscle cell the relationship between flow and MMP-2 mrna level and activity at regions of experimental arterial injury. Our working hypothesis was that low-flow conditions favor increased MMP-2 and the subsequent development of intimal thickening. To test this hypothesis, we determined MMP-2 mrna levels and activity in injured and intact New Zealand White male rabbit carotid arteries subjected to different flow conditions for 1 week. The arterial segments involved were also examined for intimal thickening. Methods Animal Model Adult New Zealand White male rabbits, weighing 3.0 to 3.5 kg and fed normal rabbit chow (n 36), were used for the study. Anesthesia was induced with injection of ketamine hydrochloride (40 mg/kg IM) and xylazine (5 mg/kg IM) and was maintained with 1% halothane via endotracheal intubation. A 2F balloon catheter was introduced twice via the facial branch of the right CCA for 10 cm to induce mural endothelial denudation and the usually associated subjacent medial injury of the entire length of the right CCA. The balloon was consistently inflated with 0.2 ml of normal saline. Injury was induced in 21 animals. Housing and handling of animals were in compliance with the Principles of Laboratory Animal Care and the Guide for the Care and Use of Laboratory Animals (NIH publication 80 23, revised 1985). Flow Modulation and Hemodynamic Measurements After balloon injury, the blood flow in the right CCA was reduced by ligation of 3 of the 4 terminal internal and external branches (n 7, low-flow group) or increased by ligation of the contralateral left CCA (n 7, high-flow group). In another group of animals with right CCA injury, flow was preserved without hemodynamic manipulation (n 7, normal-flow group). To investigate the independent roles of low and high flow, hemodynamic manipulation was undertaken in 10 other animals without balloon injury (low flow, n 5 and high flow, n 5). Five other animals served as sham-operated controls in which neither balloon injury nor flow modulation was performed (uninjured normal flow). Mean blood flow was measured with transit time ultrasound (Transonics Inc) in the right CCA before and after flow modulation. MAP was also monitored and recorded with a transduced 23-gauge indwelling catheter before and after flow modulation. The right CCA was harvested at day 7 immediately after the flow and pressure measurements. In each of the experimental groups, 4 to 5 cm of the right CCA was prepared for RNA, enzymatic analysis, and histopathological evaluation. Zymography Collagenase IV (gelatinase) activity in both injured and uninjured carotid arteries was determined by zymography. Gelatin (Sigma), 1 mg/ml, was incorporated into 8% SDS-PAGE. Samples were extracted with chilled glass homogenizers in ice-cold 10-mmol/L sodium phosphate, ph 7.2, containing 150 mmol/l sodium chloride (PBS) and 1% Triton X-100, 0.1% SDS. Samples were incubated with 0.75 mmol/l aminophenyl mercuric acetate for 30 minutes at 37 C to activate latent collagenase, 10 then mixed with sample buffer (lacking mercaptoethanol and modified to contain a final concentration of 2.5% SDS) and electrophoresed under nonreducing conditions. After electrophoresis, SDS was eluted from the gel in 2.5% Triton X-100 for 60 minutes at room temperature. The gel was then incubated in substrate buffer (50 mmol/l Tris-HCl, ph 8.0, containing 5 mmol/l CaCl 2 and 1 mol/l ZnCl 2 ) at 37 C for 16 hours. After staining with Coomassie blue R-250, gelatin-degrading enzymes were identified by their ability to clear the substrate at their respective molecular weights. Fluorometric Assay for Quantification of Gelatinase Activity Gelatinase activity was quantified by a tube assay modified from the method of Watanabe et al 15 with fluorescence gelatin as the substrate. Porcine gelatin (Sigma), 10 mg/ml, was mixed with a 20-molar excess of FITC (Sigma) in 50 mmol/l phosphate buffer, ph 9.5. The mixture was incubated at room temperature overnight and then extensively dialyzed against physiological saline for 2 to 3 days at room temperature to remove free fluorescence. The gelatin/fluorescent conjugate was diluted 1:100 (ie, to 100 g/ml) in 100 mmol/l Tris-HCl, ph 7.4, containing 5 mmol/l CaCl 2. Tissue-extracted protein (50 g in100 L) was admixed with 200 L of diluted conjugate, and the tubes were incubated at 37 C for 1 hour. After incubation, 4.0 ml of distilled water was added to all tubes to stop the reaction (by dilution), and fluorescence was quantified with excitation and emission wavelengths of 491 and 519 nm, respectively. Clostridial collagenase (Sigma) and Tris buffer were used as positive and negative controls, respectively. The amount of gelatin degraded was determined by comparison of sample readings against the positive control, which represented maximal digestion of 20 g of gelatin in the assay tube. Measurement of mrna for 72-kD Collagenase IV (MMP-2) cdna Probe A 3.1-kb cdna of human 72-kD collagenase IV (MMP-2) was obtained from Dr G.L. Goldberg, Washington University School of Medicine, St Louis, Mo. The cdna probe was recovered as a 2.0-kb fragment after digestion of the isolated plasmid with XbaI/EcoRI/ BamHI restriction enzymes. Probe was labeled with [ - 32 P]CTP by the random hexamer priming method (Promega). Total RNA Isolation and Northern Blotting All chemicals, including solvents, were of the highest analytical grade tested for negative RNAse activity and were purchased from Sigma Chemical Co. Total RNA was isolated from carotid arteries by homogenization in 4 mol/l guanidinium thiocyanate containing mercaptoethanol and extraction with phenol/chloroform. The RNA was precipitated at 70 C with isopropanol and redissolved in distilled water containing 0.1% diethylpyrocarbonate. After denaturation with formamide and formaldehyde, 15 g RNA (as measured at 260 nm) from each sample was electrophoresed in a 1.1% agarose gel at 120 V for 4 hours. Equivalency of sample loading was verified by ethidium bromide staining of the 28S and 18S rrna bands. After electrophoresis, RNA was transferred to a nylon filter by capillary blotting for 16 hours and cross-linked by ultraviolet light. The filter was then prehybridized in 50% formamide, 1 mol/l NaCl, 10 mmol/l NaH 2 PO 4,5 Denhardt s solution, 1% SDS, and 250 g/ml salmon sperm DNA at 42 C for 4 hours, followed by hybridization in the same buffer with cpm/ml of the 32 P-labeled probe. Incubation was continued overnight at 42 C. The filter was washed with 2 sodium chloride (0.15 mol/l)/sodium citrate (0.015 mol/l) (SSC) and 0.1% SDS three times for 5 minutes at room temperature, followed by two washes in 1 SSC and 0.1% SDS for 20 minutes at 50 C. The filter was subsequently exposed to x-ray film at 70 C for 72 hours. A positive control of MMP-2 transcript (3.1 kb) 16 derived from human abdominal aorta aneurysm was used to standardize the probe.

3 Bassiouny et al July 14, Figure 1. Blood flow measurements in uninjured control (U-control) and injured groups before and after flow modulation. Results are presented as mean SEM (n 5 in U-control and n 7 in injured group). *P 0.05 vs normal flow (NF) in U-control and injured group animals. Flow was similar in all six groups at baseline. After flow modulation, blood flow was markedly reduced in low-flow (LF) group and significantly increased in high-flow (HF) group compared with NF group at both time points: day 0 and day 7. Densitometric Analysis Autoradiographic bands after Northern blotting were quantified by scanning densitometry (Bio-Rad 620 scanner). To correct for differences in RNA loading, the density of the 18S ribosomal RNA band on the photographic negative of the ethidium bromide stained gel was also determined, and the relative densities of the probe and the ribosomal RNA band were compared. Histopathology The rabbits were killed with an overdose of pentobarbital, and the carotid arteries were fixed in situ by perfusion with 4% paraformaldehyde in PBS, ph 7.0, at 100 mm Hg. The arteries were embedded in paraffin, cross-sectioned at 5- m intervals, and stained with hematoxylin-eosin and Gomori s trichrome aldehyde fuchsin preparation for the connective tissue elements. When present, intimal thickening was quantified by oculomicrometry. Data Analysis Data were compared by ANOVA. A value of P 0.05 was considered to be significant. Data are expressed as mean SEM. Results Hemodynamic Parameters Mean baseline blood flow (21 ml/min) was similar before flow modulation in all groups and ranged from 19 to 22 ml/min. In the low-flow animals, outflow ligation reduced baseline flow 3-fold at day 0, to 7 1 ml/min (injured) and 6 1 ml/min (uninjured control) (P 0.05). In the high-flow animals, ligation of the contralateral left CCA increased baseline flow by 50% at day 0, to 31 1 ml/min (injured) and 35 2 ml/min (uninjured control) (P 0.05). In the normalflow animals, blood flow was unchanged at day 0 compared with the baseline flow. At day 7 before death, the blood flow measurements in each group were not different from the initial values recorded on day 0 (Figure 1). MAP in the right CCA was not different between the low-, high-, or normal-flow groups before and after flow modulation. At day 7, the MAPs in each group were maintained at the day 0 levels, showing that despite significant flow changes, the MAPs did not change from the baseline values (Figure 2). Gelatinolytic Activity Zymography of the arterial extracts demonstrated a consistent band of gelatinolytic activity at the molecular size of 72 kd, representing gelatinase A (MMP-2). This was constitutively expressed in uninjured rabbit carotid arteries and was increased at 7 days after arterial injury in all experimental groups. Zones of lysis were also observed at molecular positions of 64, 80, and 90 kd (Figure 3). The 72-kD MMP-2 and its activated 64-kD form were greatest in the low-flow group in both injured and uninjured arteries than in the normal-flow and high-flow groups. Lesser-intensity bands at 90 and 80 kd appeared only in the injured low-flow group (not discernible in Figure 3). These most likely represent 92-kD collagenase IV (MMP-9) and its activated forms. 8 As expected from the zymogram in Figure 3, quantitative gelatinase activity (ng gelatin digested h 1 mg protein 1 ) was significantly increased at 1 week in both injured (50 5) and uninjured (48 3) right CCA segments subjected to low flow compared with the respective injured (29 2) and uninjured (26 4) segments of normal flow (P 0.05). Although moderate increases in activity were also noted in high-flow conditions, 41 4 for uninjured and 35 3 for injured segments, this was not statistically different from the normal-flow groups (Figure 4). MMP-2 mrna Expression To determine whether flow induced alteration of MMP-2 mrna, we performed Northern blot analysis. Rabbit MMP-2 Figure 2. MAP measurements in uninjured control (U-control) and injured groups before and after flow modulation. Results are presented as mean SEM (n 5 in U-control and n 7 in injured group). No significant differences were observed before and after flow modulation in all three flow groups.

4 160 Flow Regulation of MMP-2 Figure 3. Zymographic analyses of gelatinases from carotid artery extracts in normal-flow (NF), low-flow (LF), and high-flow (HF) groups from uninjured control (U) and injured (I) animals. Each result is shown as duplicate lanes. Zymogram shows increased activity of 72-kD gelatinase and its activated form (64 kd) in LF uninjured and injured arteries vs NF and HF. Activity was greater in injured arteries in NF and HF than uninjured arteries. mrna was represented by two species of mrna: 2.8 and 3.5 kb. The detection of these two transcripts with a human MMP-2 cdna probe is consistent with recent work by Matsumoto et al, 17 in which 95% homology was shown between human and rabbit MMP-2. Overall, MMP-2 mrna expression was upregulated in the injured arterial segments compared with uninjured normal-flow segments, as represented in Figure 5A. RNA loading conditions in each lane were assessed with 18S rrna. Relative mrna levels for each group were determined by densitometry using both bands as signals and normalizing the density to 18S rrna and were expressed as a percentage increase of the uninjured normal-flow values (Figure 5B). There was a 186% increase in MMP-2 mrna levels in injured arterial segments with normal flow (P 0.05) and a 366% increase with low flow (P 0.005). Conversely, mrna levels were only slightly increased (38%) with high flow (P 0.05). Within the injured arterial segments, MMP-2 mrna was 2-fold greater with low flow (P 0.05) and less then half with high flow (P NS) compared with normal flow. In uninjured arterial segments, MMP-2 mrna levels were increased by 39% in the low-flow (P 0.05) and 26% in the high-flow (P 0.05) groups compared with normal-flow control values (Figure 6A and 6B). Histopathology Histological sections of the right CCA from each experimental group were examined for intimal thickening. This was Figure 4. Quantification of gelatinase activity from uninjured control (U-control) and injured carotid arteries under different flow conditions after 7 days. Results are presented as mean SEM (n 5 in U-control and n 4 in injured group). *P 0.05 vs normal flow (NF) in U-control and injured animals, respectively. Gelatinase activity increased 50% in low-flow (LF) group vs NF group. There was also a moderate increase in activity in high-flow (HF) group. This was not statistically different from NF group.

5 Bassiouny et al July 14, Figure 5. A, Northern blot analysis for 72-kD gelatinase (MMP-2) mrna levels in injured carotid arteries. Each result is presented in duplicate lanes. Results from uninjured normal-flow carotid arterial segments (U-NF) are shown for comparison. NF indicates normal flow; LF, low flow; and HF, high flow. Ethidium bromide staining of 18S rrna bands indicates loading conditions. B, Densitometric analysis of blot. Densities were compared in each case (n 4) with U-NF bands. MMP-2 mrna at 3.5 and 2.8 kb was increased in all injured carotid segments (NF, LF, and HF) vs U-NF. Increase in LF group was greatest (366%, P 0.005), in NF moderate (186%, P 0.05), and in HF minimal (38%, P 0.05). Within injured arterial segments, MMP-2 mrna was 2-fold greater with low flow (P 0.05, ) and less than half with high flow (P NS) vs normal flow. observed only in the injured arteries subjected to low-flow conditions (n 3). At 1 week, intimal thickening varied between 0.02 and 0.04 mm. The lesion was composed of SMCs overlying the internal elastic lamina, with no evidence of mural thrombosis (Figure 7). Figure 6. A, Northern blot analysis for 72-kD gelatinase (MMP-2) mrna levels in uninjured carotid arteries. Each result is presented in duplicate lanes. NF indicates normal flow; LF, low flow; and HF, high flow. Ethidium bromide staining of 18S rrna bands indicates loading conditions. B, Densitometric analysis of blot. Densities were compared in each case (n 5) with NF bands. MMP-2 mrna at 3.5 and 2.8 kb was increased by 39% (P 0.05) in LF and 26% (P 0.05) in HF, respectively, vs NF carotid segments. Discussion The findings of this study indicate that hemodynamic forces may modulate in vivo MMP-2 transcription and activity in the rabbit carotid artery and substantiate the paradigm that low-flow and shear conditions enhance intimal thickening through mechanisms that regulate extracellular matrix degradation. The latter is a critical step if the proliferating SMCs were to migrate to the subendothelial space. In this investigation, we examined the independent role of flow and injury in relation to MMP-2 mrna levels and its gelatinolytic activity. We found that arterial mural injury upregulated MMP-2 gene expression at 7 days. However, this was significantly enhanced by low-flow and, conversely, inhibited by relatively high-flow conditions. Concomitantly, gelatin lysis by 72-kD gelatinase and its activated form 64-kD gelatinase was remarkably increased in low-flow conditions compared with control uninjured arteries and injured arteries with normaland high-flow conditions. This increase in gelatinolytic activity was also confirmed by fluorometric gelatinase activity assay. Intimal thickening was observed only in those arterial segments subjected to injury and low-flow conditions. Other investigators have shown that MMPs are involved in the migration and proliferation of vascular SMCs in culture and in experimental arterial injury. In a recent study by Pauly et al, 10 isolated rat VSMCs cultured on a gel of reconstituted basement membrane proteins secreted an active form of MMP-2 to degrade complex extracellular matrix proteins. Neutralizing antisera to MMP-2 inhibited VSMC migration through the basement membrane barrier, indicating that VSMC migration may be dependent on MMP-2 activity. Southgate et al 8 demonstrated that two synthetic MMP inhibitors specific to MMP-2 and MMP-9 inhibited proliferation of SMCs from rabbit aortic explants in a concentrationdependent manner. In the rat carotid artery, there is constitutive expression of MMP-2 with increased activity between 4 and 14 days after injury, whereas MMP-9 was induced the first day after injury. 3,6 The early migration of SMCs into the intima was reduced 97% when an MMP inhibitor was administered. 3,11 A similar pattern was also found in the porcine carotid artery after balloon injury. Increased gene expression of MMP-2 and MMP-9 was found in both intimal and medial SMCs and around foci of medial necrosis. 18 The role of flow and related shear-mediated forces in modulating these characteristic MMP responses to in vivo arterial injury has not been demonstrated previously. Our findings indicate that when injured arteries are exposed to

6 162 Flow Regulation of MMP-2 Figure 7. Morphological changes in carotid artery of three experimental groups on day 7 after injury. Intimal thickening developed only in low-flow group (A). Lesion was composed of SMCs overlying internal elastic lamina (IEL) with no evidence of mural thrombosis. Intimal thickening was not observed in normal-flow (B) or high-flow (C) group. Adv indicates adventitia. low- or high-flow conditions, a differential response in MMP-2 activity and relative mrna levels is observed, with upregulation by low flow and downregulation by high flow. It should be emphasized that the blood pressure parameters between the three flow groups were similar, thus controlling for the potential confounding variable of tensile stress, which has been shown to greatly modify SMC metabolism. Another important objective of this investigation was to further elucidate the independent role of flow on MMP-2 transcription and activity. Interestingly, increased gelatinase activity was evident and of similar order of magnitude in low-flow arterial segments without and with injury. Conversely, MMP-2 mrna showed a less dramatic increase in low-flow uninjured tissue compared with the injured group. This suggests that increased MMP-2 activity is induced by low flow independent of endothelial and SMC injury. We have previously demonstrated that two types of intimal hyperplasia are found in experimental anastomotic regions: suture line intimal thickening, which represents vascular healing, and arterial floor intimal thickening, which developed in regions of flow oscillation and relatively low shear. 12 Increasing flow by creating a distal arteriovenous fistula markedly reduced anastomotic intimal thickening in experimental end-to-side polytetrafluoroethylene anastomoses. 19 Low shear stress was also found to promote intimal hyperplasia and cellular proliferation in vein and endothelialized prosthetic grafts 20,21 and in injured rat carotid arteries. 22 Varying shear stress also appears to modulate SMC proliferation in vitro. Sterpetti et al 23 reported that increasing shear stress inhibits and decreasing shear stress facilitates proliferation of bovine arterial SMCs. This response was associated with enhanced PDGF expression. 20,21,23 PDGF is a potent mitogen for SMCs and a stimulator for MMPs. It is secreted not only by platelets but also by endothelial cells, SMCs, and macrophages. Because a putative shear stress responsive element (GAGACC) has been identified in the 5 -flanking region of the PDGF-B gene, 24 low-flow and shear-induced MMP-2 activity may therefore be related indirectly to shearmediated PDGF secretion from SMCs. Another potential mechanism for flow modulation of MMP-2 is via platelet activation during injury. Preliminary findings in our laboratory suggest that platelet activation as measured by thromboxane B 2 levels is greater after experimental arterial injury with injured arteries subjected to reduced flow compared with normal and increased flow conditions. This suggests that platelet adhesion to regions of injury is enhanced in a low-flow and low-shear environment. Moreover, activated platelets have recently been found to release MMP-2, which mediates further platelet aggregation. 25 Activated platelets release many growth factors, including PDGF, basic fibroblast growth factor, and transforming growth factor. These factors play an important role in regulating SMC migration and proliferation 29,30 and may also induce MMP expression during arterial injury. 4,31 Conversion of MMP latent proenzyme to its active form by the plasminogen activator system is also enhanced by growth factors such as PDGF and basic fibroblast growth factor. 32 Thus, in this experimental model, regulation of MMPs by flow may occur at two levels, transcriptional and activation of the latent proenzyme. Hemodynamic shear forces induce in vivo MMP-2 transcriptional events that are independent of yet enhanced by injury. Upregulation of collagenase activity is observed with low flow and is independent of mural injury. Potential mechanisms for this relationship include regulation of MMP-2 release from shear-activated SMCs or platelets or indirectly via cytokine-mediated pathways. Acknowledgments This work was supported by American Heart Association Grant-in- Aid The authors would like to thank Xiling Liu, biostatistician, for the data analysis and James Vosicky for his excellent assistance in operative and postoperative animal care.

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